The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.